Ketohexokinase-C: The mechanistic link between dietary sugars and protein acetylation during liver steatosis
Authors: Naiara Beraza
Journal: Journal of Hepatology (2023)

Ketohexokinase-C regulates global protein acetylation to decrease carnitine palmitoyltransferase 1a-mediated fatty acid oxidationJournal of HepatologyVol. 79Issue 1PreviewThe consumption of sugar and a high-fat diet (HFD) promotes the development of obesity and metabolic dysfunction. Despite their well-known synergy, the mechanisms by which sugar worsens the outcomes associated with a HFD are largely elusive. Full-Text PDF See Article, pages 25–42 See Article, pages 25–42 Non-alcoholic fatty liver disease (NAFLD) affects around 25% of the Western world’s population1Williams R. Aspinall R. Bellis M. Camps-Walsh G. Cramp M. Dhawan A. et al.Addressing liver disease in the UK: a blueprint for attaining excellence in health care and reducing premature mortality from lifestyle issues of excess consumption of alcohol, obesity, and viral hepatitis.Lancet. 2014; 384: 1953-1997Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar, 2Abeysekera K.W.M. Fernandes G.S. Hammerton G. Portal A.J. Gordon F.H. Heron J. et al.Prevalence of steatosis and fibrosis in young adults in the UK: a population-based study.Lancet Gastroenterol Hepatol. 2020; 5: 295-305Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 3Younossi Z.M. Koenig A.B. Abdelatif D. Fazel Y. Henry L. Wymer M. Global epidemiology of nonalcoholic fatty liver disease-Meta-analytic assessment of prevalence, incidence, and outcomes.Hepatology. 2016; 64: 73-84Crossref PubMed Scopus (5863) Google Scholar and is the most common chronic liver disease worldwide.[4]Powell E.E. Wong V.W. Rinella M. Non-alcoholic fatty liver disease.Lancet. 2021; 397: 2212-2224Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar The term NAFLD covers a series of pathological disorders including non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH) that can progress to fibrosis, cirrhosis and ultimately hepatocellular carcinoma.[4]Powell E.E. Wong V.W. Rinella M. Non-alcoholic fatty liver disease.Lancet. 2021; 397: 2212-2224Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar NAFLD associates with comorbidities that include obesity, type 2 diabetes and cardiovascular disease, the latter being the leading cause of death in patients with NAFLD.[4]Powell E.E. Wong V.W. Rinella M. Non-alcoholic fatty liver disease.Lancet. 2021; 397: 2212-2224Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar (Fig. 1) A sedentary lifestyle and high-calorie Western diets contribute to the development and progression of NAFLD. Poor dietary habits commonly combine high-fat with high-sugar intake, often including consumption of sugar-sweetened beverages that contain fructose, glucose or sucrose (50% fructose, 50% glucose). Several studies have established the significant association between the risk of developing NAFLD and consumption of sugar-sweetened beverages,[5]Ma J. Fox C.S. Jacques P.F. Speliotes E.K. Hoffmann U. Smith C.E. et al.Sugar-sweetened beverage, diet soda, and fatty liver disease in the Framingham Heart Study cohorts.J Hepatol. 2015; 63: 462-469Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar,[6]Yki-Jarvinen H. Luukkonen P.K. Hodson L. Moore J.B. Dietary carbohydrates and fats in nonalcoholic fatty liver disease.Nat Rev Gastroenterol Hepatol. 2021; 18: 770-786Crossref PubMed Scopus (65) Google Scholar which contribute to steatosis by promoting de novo lipogenesis (DNL) in the liver. Fructose is a particularly potent activator of DNL,[5]Ma J. Fox C.S. Jacques P.F. Speliotes E.K. Hoffmann U. Smith C.E. et al.Sugar-sweetened beverage, diet soda, and fatty liver disease in the Framingham Heart Study cohorts.J Hepatol. 2015; 63: 462-469Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar,[7]Ouyang X. Cirillo P. Sautin Y. McCall S. Bruchette J.L. Diehl A.M. et al.Fructose consumption as a risk factor for non-alcoholic fatty liver disease.J Hepatol. 2008; 48: 993-999Abstract Full Text Full Text PDF PubMed Scopus (608) Google Scholar via the activation of lipogenic genes like SREBP1-c and ChREBP. During DNL, there is also an increase in malonyl-CoA which inhibits carnitine palmitoyltransferase 1A (CPT1α), resulting in inhibition of mitochondrial fatty acid oxidation (FAO) and lipid accumulation in the liver.[8]McGarry J.D. Mannaerts G.P. Foster D.W. A possible role for malonyl-CoA in the regulation of hepatic fatty acid oxidation and ketogenesis.J Clin Invest. 1977; 60: 265-270Crossref PubMed Google Scholar ATP-citrate lyase also plays a role in mediating the increase in malonyl-CoA by converting citrate into acetyl-CoA.[9]Postic C. Girard J. Contribution of de novo fatty acid synthesis to hepatic steatosis and insulin resistance: lessons from genetically engineered mice.J Clin Invest. 2008; 118: 829-838Crossref PubMed Scopus (923) Google Scholar In a previous study, Softic et al. proposed that increases in lipid accumulation in the liver in response to consumption of fructose combined with high-fat diets resulted not only from elevated fatty acid synthesis, but also from a reduction in FAO capacity associated with increased malonyl-CoA, changes in mitochondrial size and acetylation of CPT1α.[10]Softic S. Meyer J.G. Wang G.X. Gupta M.K. Batista T.M. Lauritzen H. et al.Dietary sugars alter hepatic fatty acid oxidation via transcriptional and post-translational modifications of mitochondrial proteins.Cell Metab. 2019; 30: 735-753 e734Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar Interestingly, these effects were not fully recapitulated by glucose supplementation, supporting the detrimental contribution of fructose in combination with a high-fat diet to liver steatosis. These observations agree with the more profound decrease in FAO observed in individuals after fructose consumption than in those consuming glucose.[11]Cox C.L. Stanhope K.L. Schwarz J.M. Graham J.L. Hatcher B. Griffen S.C. et al.Consumption of fructose-sweetened beverages for 10 weeks reduces net fat oxidation and energy expenditure in overweight/obese men and women.Eur J Clin Nutr. 2012; 66: 201-208Crossref PubMed Scopus (108) Google Scholar In this issue of Journal of Hepatology, Softic et al. further investigate the mechanisms mediating the synergic effect of dietary sugar and fat on NAFLD and report that ketohexokinase-C (KHK-C) is a mediator of liver lipid accumulation, via a reduction of FAO and global protein acetylation. Fructose is metabolised in the liver by phosphorylation to fructose-1-phosphate via KHK-C, a reaction not regulated by insulin. KHK-C expression is increased in the liver of patients with NAFLD who consume fructose, and in vitro studies confirmed the dose-dependent effect of fructose in upregulating KHK-C.[7]Ouyang X. Cirillo P. Sautin Y. McCall S. Bruchette J.L. Diehl A.M. et al.Fructose consumption as a risk factor for non-alcoholic fatty liver disease.J Hepatol. 2008; 48: 993-999Abstract Full Text Full Text PDF PubMed Scopus (608) Google Scholar Inhibition of KHK-C in high-fat diet (HFD)-fed mice supplemented with fructose led to reduced fatty acid synthesis and improved liver steatosis,[12]Softic S. Gupta M.K. Wang G.X. Fujisaka S. O'Neill B.T. Rao T.N. et al.Divergent effects of glucose and fructose on hepatic lipogenesis and insulin signaling.J Clin Invest. 2017; 127: 4059-4074Crossref PubMed Scopus (180) Google Scholar providing evidence in support of the lipogenic activity of fructose. Now, Softic et al. show that both fructose consumption in chow-fed mice and fructose and glucose supplementation of HFD resulted in elevated expression of KHK-C. The authors propose that upregulation of KHK-C following glucose supplementation of HFD-fed mice resulted from endogenous fructose production in response to glucose. Interestingly, increased KHK-C was associated with elevated expression of lipogenic proteins, while their mRNA expression remained unaffected, as did activation of lipogenic transcription factors SREBP-1c and ChREBP, suggesting that KHK-C controls lipogenic enzymes post-translationally, particularly ATP-citrate lyase. Also, while other intermediates of DNL and the tricarboxylic acid cycle remained largely unaffected by dietary sugar, increases in KHK-C were associated with changes in mitochondrial size, particularly in HFD-mice supplemented with fructose. Based on these results, Softic et al. proposed that increased fructolysis reduces mitochondrial FAO; thus, they examined the expression of CPT1α, the rate-limiting enzyme controlling FAO. The authors found that the increase in KHK-C in sugar-supplemented mice was associated with a reduction in CPT1α protein expression, particularly when combined with a HFD. The inverse correlation between KHK-C and CPT1α was confirmed in alternative in vivo models of NAFLD, including genetically obese db/db mice and lipodystrophic FIRKO (fat-specific insulin receptor knockout) mice, and in vitro in AML12 hepatocytes overexpressing KHK-C. The reduction in CPT1α after overexpression of KHK-C in vitro led to a metabolic switch characterised by decreased FAO and increased glycolysis. When treated with free fatty acids, cells over-expressing KHK-C showed an elevated accumulation of triglycerides. Expression of CPT1α can be controlled transcriptionally and post-translationally by malonyl-CoA.[8]McGarry J.D. Mannaerts G.P. Foster D.W. A possible role for malonyl-CoA in the regulation of hepatic fatty acid oxidation and ketogenesis.J Clin Invest. 1977; 60: 265-270Crossref PubMed Google Scholar Interestingly, the authors found that reduction in CPT1α protein expression in sugar-supplemented HFD-fed mice was not correlated with changes in RNA expression or malonyl-CoA. Remarkably, KHK-C overexpression led to an increase in global protein acetylation which affected acyl-CoA metabolism, FAO-associated proteins and promoted acetylation of CPT1α at K508, which reduced protein expression. Protein acetylation is mainly regulated by deacetylases. Sirtuins are a family of NAD+-dependent histone III deacetylases that tightly regulate gene and protein expression. Sirtuins play key roles in regulating cell metabolism, including by promoting mitochondrial biogenesis and FAO, as well as by restricting lipogenesis via deacetylation.[13]Houtkooper R.H. Pirinen E. Auwerx J. Sirtuins as regulators of metabolism and healthspan.Nat Rev Mol Cell Biol. 2012; 13: 225-238Crossref PubMed Scopus (497) Google Scholar In the context of increased global acetylation, Softic et al. showed that while the majority of sirtuins were unaffected by KHK-C overexpression, there was a reduction in SIRT2 in mice receiving fructose and a HFD, pointing to regulation of this deacetylase as a mechanistic mediator of KHK-C-induced global acetylation, specifically of CPT1α. Previous studies reporting a reduced presence of SIRT2 during NAFLD[14]Ren H. Hu F. Wang D. Kang X. Feng X. Zhang L. et al.Sirtuin 2 prevents liver steatosis and metabolic disorders by deacetylation of hepatocyte nuclear factor 4alpha.Hepatology. 2021; 74: 723-740Crossref PubMed Scopus (23) Google Scholar support a role for this deacetylase in mediating KHK-C-associated increases in lipid accumulation via control of protein acetylation. Overall, the work of Softic et al. describes a novel role for KHK-C in contributing to liver lipid accumulation in response to dietary sugars via the control of protein acetylation, which leads to stabilisation of DNL proteins and a reduction of FAO as a result of reduced CPT1α protein expression (Fig. 1). Currently there are no licenced pharmacological treatments for NAFLD and changes in dietary habits and lifestyle are the only treatment options. Clinical trials using drugs to target NAFLD have shown limited results and there is thus a need to develop new therapeutics. Previous studies showing that KHK was elevated in individuals with NAFLD[7]Ouyang X. Cirillo P. Sautin Y. McCall S. Bruchette J.L. Diehl A.M. et al.Fructose consumption as a risk factor for non-alcoholic fatty liver disease.J Hepatol. 2008; 48: 993-999Abstract Full Text Full Text PDF PubMed Scopus (608) Google Scholar led to the development of small molecule inhibitors of the kinase activity of KHK as potential treatments for NAFLD,[15]Futatsugi K. Smith A.C. Tu M. Raymer B. Ahn K. Coffey S.B. et al.Discovery of PF-06835919: a potent inhibitor of ketohexokinase (KHK) for the treatment of metabolic disorders driven by the overconsumption of fructose.J Med Chem. 2020; 63: 13546-13560Crossref PubMed Scopus (21) Google Scholar though their use has recently been discontinued in several clinical trials. Based on their findings, Softic et al. propose that inhibitors of the kinase activity of KHK would only target enzymatic regulation of DNL, while KHK-C protein acetylation-associated inhibition of CPT1α/FAO would remain intact, resulting in limited efficacy. Thus, the novel regulatory capacity of KHK-C to regulate protein acetylation may open new avenues for development of therapeutics based on complete, rather than partial, inhibition of KHK activity. The efficacy of this approach would be supported by preclinical studies demonstrating efficacy of (complete) genetic depletion of KHK-C in reducing liver steatosis.[16]Shepherd E.L. Saborano R. Northall E. Matsuda K. Ogino H. Yashiro H. et al.Ketohexokinase inhibition improves NASH by reducing fructose-induced steatosis and fibrogenesis.JHEP Rep. 2021; 3100217PubMed Google Scholar However, such an approach targeting a single molecule may have limitations based on the complexity of NAFLD, which not only involves different organs (adipose, intestine, liver, brain), but also the immune system and the gut microbiome. This would argue against a ‘magic bullet’ treatment for all aspects of NAFLD, while single drug approaches may still benefit specific aspects of the disease. Another consideration regarding the translational significance of the Softic et al. study is how relevant it is to adult populations, as the results shown were obtained in young male mice (6 weeks-old), closer to adolescence than to adulthood. Since the global burden of NAFLD is high in this age-group, this does not curtail the significance of the study, but it is relevant to its applicability for the wider population. Another limitation in many preclinical studies is the use of single sex mice (generally males, as in this study), particularly in the context of NALFD where there are metabolic differences between the sexes. While the prevalence of NAFLD is lower in females than males, this apparent protection is drastically reduced in postmenopausal females, suggesting oestrogen protects against NALFD development. Mechanistically, oestrogen replacement therapy (with oestradiol) reduced DNL in ob/ob mice[17]Gao H. Bryzgalova G. Hedman E. Khan A. Efendic S. Gustafsson J.A. et al.Long-term administration of estradiol decreases expression of hepatic lipogenic genes and improves insulin sensitivity in ob/ob mice: a possible mechanism is through direct regulation of signal transducer and activator of transcription 3.Mol Endocrinol. 2006; 20: 1287-1299Crossref PubMed Scopus (142) Google Scholar and restored FAO in livers from mice that lack intrinsic oestrogen production.[18]Nemoto Y. Toda K. Ono M. Fujikawa-Adachi K. Saibara T. Onishi S. et al.Altered expression of fatty acid-metabolizing enzymes in aromatase-deficient mice.J Clin Invest. 2000; 105: 1819-1825Crossref PubMed Google Scholar Accordingly, men have higher postprandial DNL,[19]Pramfalk C. Pavlides M. Banerjee R. McNeil C.A. Neubauer S. Karpe F. et al.Sex-specific differences in hepatic fat oxidation and synthesis may explain the higher propensity for NAFLD in men.J Clin Endocrinol Metab. 2015; 100: 4425-4433Crossref PubMed Scopus (94) Google Scholar supporting sex differences in regulating liver lipid metabolism in response to dietary sugars. Hence, it would be important to investigate whether the regulation of lipid metabolism reported by Softic and colleagues also occurs in females, as well as the potential interactions between oestrogen and KHK-C in controlling liver lipid accumulation in females. Overall, studies like the elegant work of Softic et al. contribute to improving our fundamental knowledge and understanding of the precise mechanisms mediating progression of NAFLD. Studies of this nature are essential to collectively enable the development of therapeutics targeting different pathways to tackle progression of NAFLD. NB is funded by the BBSRC Institute Strategic Programme Gut Microbes and Health BBS/E/F/00044509, the BBSRC Gut Microbes and Health Institute Strategic Programme BB/R012490/1 and its constituent project BBS/E/F/000PR10355, and the BBSRC Core Capability Grant BB/CCG1860/1. NB has no competing financial interests with respect to this manuscript. Please refer to the accompanying ICMJE disclosure forms for further details. The following are the supplementary data to this article: Download .pdf (.13 MB) Help with pdf files Multimedia component 1